The gel microdrop (GMD) secretion assay involves encapsulating cells in a biotinylated matrix, followed by capture and detection of cell-secreted molecules with fluorescent markers (17-34). This technology differs from other encapsulation methods in that the small size of the microdrop (e.g.,  less than 50 xcexcm diameter) creates a defined microenvironment around the cell without impeding diffusion of nutrients, antibodies, or nucleic acid probes into the microdrops, or diffusion of secreted products. Furthermore, microdrops can readily be analyzed using flow cytometry and sub-populations can be detected. The number of occupied cells in each microdrop preparation depends on the number of cells used for encapsulation and is approximated by Poisson statistics for single cell encapsulation (19). To obtain microdrops having a high probability of initially containing 0 or 1 cells, an experimental protocol has been developed in which 1-1.5 million cells are encapsulated in 20 million microdrops, resulting in approximately 5-10% single cell occupation . . . . The emulsion is transiently cooled, causing the drops to gel. Once gelled, the microdrops are physically distinct and robust and can be removed from the oil into an aqueous medium by low speed centrifugal separation. Since the microdrop agarose matrix is a permeable semi-solid support, immunochemical procedures can be performed on encapsulated cells.
The invention provides methods of analzying a secreted protein. Such method entail encapsulating a cell in a microdrop wherein the microdrop comprises matrix component molecules, first biotin molecules linked to the matrix component molecules, capture molecules with affinity for a molecule secreted by the cell linked to second biotin molecules, and streptavidin linking the first and second biotin molecules, the first biotin molecules and the matrix component molecules being in a molar ratio of less than 0.85, preferably 0.01 to 0.2, and optionally 0.02-0.2 moles biotin per mole matrix component molecules. The molecule is secreted from the cell and binds to the capture molecules is thereby retained within the microdrop. The secreted molecule is then detected. In some methods the concentration of the first biotin molecules in the microdrop is less than or equal to 42 micromolar.
In some methods, the encapsulating step encapsulates a plurality of cells in the microdrop. In some methods, the encapsulating step encapsulates a single cell in the microdrop. In some methods, the secreted molecule is a protein, hormone, or carbohydrate. In some methods, the streptavidin and capture molecules are encapsulated into the microdrops at the same time as the cell. In some methods, the streptavidin and captured molecules are incorporated into the microdrop after the encapsulating step. In some methods, the microdrop further comprises second capture molecules with affinity for a second secreted molecule, the second capture molecules being linked to additional copies of the second biotin molecules. In some methods, the cell secretes the second molecule. In some methods, the detecting step is performed by contacting the microdrop with a detection reagent having affinity for the secreted molecule, and detecting binding of the detection reagent to the secreted molecule. In some methods, the detection reagent is labeled. In some methods, a signal of the labeled detection reagent bound to the secreted molecule is proportional to the number of copies of the secreted molecule within the microdrop. In some methods, the detecting step is performed by contacting the microdrop with a first detection reagent having specific affinity for the secreted molecule and a second detection reagent having specific affinity for the second secreted molecule, wherein the first and second detection reagents are differentially labeled. In some methods, the cell secretes a third secreted molecule, and the microdrop further comprises third capture molecules with affinity for the third secreted molecule, the third capture molecules being linked to additional copies of the second biotin molecules, and the method further comprises contacting the microdrop with first, second, third and fourth detection reagents having specific affinity for the secreted molecule, the second secreted molecule, the third secreted molecule and a cell surface marker respectively, and the detecting step detects the secreted protein, the second secreted protein, the third secreted protein and the cell surface marker. In some methods, the detection step is performed by contacting the microdrop with a first detection reagent having affinity for the secreted molecule and a second detection reagent having affinity for a cell surface marker, and the first and second detection reagents are differentially labeled. In some methods, the detection reagent and the capture molecules bind to different epitopes on the secreted molecule. In some methods, the matrix component is agarose. In some methods, the secreted protein is an antibody. In some methods, the secreted protein is an antibody of IgG isotype and the capture molecules are antibodies specific for the IgG isotype. In some methods, the secreted protein is a cytokine. Some methods further comprise inducing the cell to secrete the secreted molecule. In some methods, the inducing is performed after the encapsulating step. In some methods, the inducing is performed before the encapsulating step.
In some methods, the cell comprises a vector comprising a nucleic acid segment encoding the secreted protein, the segment being operably linked to one or more regulatory DNA segments that effect expression of the secreted protein. In some methods, the secreted protein is naturally secreted by the cell. Some methods further comprise a step of propagating the cell to form a cell line after the detecting step. In some methods, the cell was obtained from a patient. In some methods, the cell is a cytotoxic T-cell. Some methods further comprise introducing a population of cells resulting from propagating the cell into the patient. In some methods, the cell is a stem cell. In some methods, the patient is suffering from an autoimmune disease and the cell is a Th2 cell. In some methods, the cell is an islet cell secreting insulin and the patient is in a prodromal period prior to onset of clinical symptoms. Some methods further comprise treating the cell with IL-10 during the propagating step. Some methods further comprise separating the cells from other cells using a cell sorter based on a fluorescent signal resulting from binding of a fluorescently labeled detection reagent to the secreted molecule. In some methods, the cell secretes first and second proteins, and the matrix comprises first and second capture molecules with affinity for the first and second proteins respectively, and the analyzing step comprises contacting the cell with first and second detection reagents that bind to the first and second secreted proteins, and detecting the first and second proteins from signal of the first and second detecting reagents bound to the first and second secreted proteins.
The invention provides methods of analyzing a population of cells. Such methods entail encapsulating a population of cells in microdrops. The cells are contacted with a first detection reagent for a first marker, and a second detection reagent for a second marker, wherein the first marker is a secreted protein and the second marker is a cell surface protein or a second secreted protein. Cells having both the first and second markers are detected. In some methods, at least some microdrops encapsulate single cells. Some methods further comprise separating the microdrops encapsulating the cells from unoccupied microdrops on a Percoll, polysucrose, sodium diatrizoate, or iodixanol gradient. Some method further comprise forming an array of microdrops encapsulating the cells attached to a solid support, and wherein the detecting is performed using a scanning fluorescent, colorimetric, chemiluminescent detector. Some methods further comprise contacting the microdrops encapsulating cells with an agent, and wherein the detecting indicates whether the agent affects the level of the secreted protein.
In some methods, the encapsulated cells are population of cells from a patient and the agent is a different population of cells from the patient. In some methods, the sub-population of cells is isolated by binding of fluorescently labelled antibody to a cell surface marker, and detection of the fluorescent signal. In some methods, the population of cells are obtained from a patient and the presence of the cells having both the first and second markers indicates an immune status of the patient. In some methods, the cells are antigen-specific T-cells. In some methods, the first and second markers are selected from the group consisting of IL-4, IL-10, IFNgamma and TNFalpha. In some methods, the first and second particular proteins are selected from the group consisting of IL-10 and IL-12. In some methods, the identified cells are Th1 cells. In some methods, the identified cells are Th2 cells. In some methods, the second marker is a cell surface marker of T-cell differentiation. In some methods, one of the markers is CD4 or CD8.
The invention further provides methods of analyzing a population of cells. Such methods entail encapsulating a population of cells expressing proteins in microdrops, and wherein the microdrops comprise matrix component molecules, first biotin molecules linked to matrix component molecules, at least first capture molecules having affinity for an epitope in at least one type of secreted protein; the first capture molecules being linked to second biotin molecules, and streptavidin, linking the first and second biotin molecules; whereby proteins are secreted from the cells and proteins having affinity for the first capture molecules are captured within the microdrops; and analyzing the secreted proteins within the microdrops. In some methods, at least some of the microdrops encapsulate a single cell. In some methods, the cells secrete the same protein at different levels, and the analyzing step compares the levels. In some methods, the cells secrete different proteins, and the analyzing step detecting a cell secreting a particular protein. In some methods, the population of cells was obtained from the patient, and the analyzing identifies a subpopulation of cytotoxic T-cells and the method further comprises reintroducing the population of cells without the subpopulation of cells into the patient. In some methods, the patient is suffering from or susceptible to an autoimmune disease, graft versus host disease or host versus graft disease. Some methods further comprise treating the population of cells without the subpopulation of cells with IL-10 before the reintroducing step.
The invention further provides a population of microdrops encapsulating cells, and the microdrops comprise matrix component molecules, first biotin molecules linked to the matrix component molecules, capture molecules with affinity for a protein secreted by the cell linked to second biotin molecules, and streptavidin linking the first and second biotin molecules, the first biotin molecules and the matrix molecules being in a molar ratio of less than 0.85, preferably less than 0.01-0.2, and optionally less than 0.02-0.2 moles biotin per mole matrix component molecules. In some populations at least some microdrops encapsulate a single cell.
The invention further provides in a method of analyzing a protein secreted by a cell in which the cell is encapsulated with a microdrop comprising biotinylated agarose the improvement wherein the molar ratio of biotin to agarose is less than 0.85, preferably 0.01 to 0.2, and optionally 0.02 to 0.2 moles biotin per mole agarose.
The invention further provides methods of analyzing a secreted protein. Such methods entail encapsulating a cell in a microdrop wherein the microdrop comprises matrix component molecules, Ni2+NTA linked to the matrix component molecules, capture molecules with affinity for a molecule secreted by the cell linked to a hexahistidine tag, wherein the molecule is secreted from the cell and binds to the capture molecules thereby being retained within the microdrop; and contacting the cell with a detection reagent that binds to the secreted molecule wherein the detection reagent.
The method further provides methods of analyzing a secreted protein. Such method entail encapsulating a cell in a microdrop wherein the microdrop comprises matrix component molecules linked to biotin, streptavidin linked to Ni2+ and capture molecules with affinity for a molecule secreted by the cell linked to a hexahistidine tag, wherein the molecule is secreted from the cell and binds to the capture molecules thereby being retained within the microdrop; and contacting the cell with a detection reagent that binds to the secreted molecule wherein the detection reagent.
The invention further provides method s of preparing an antibody of IgG isotype. Such methods entail culturing a population of cells secreting antibodies of IgM isotype under conditions whereby one or more of the cells can undergo isotype switching to IgG isotype; encapsulating the population of cells in microdrops including a capture reagent specific for antibodies of IgG isotype, whereby microdrops containing a cell secreting an antibody of IgG isotype capture the secreted antibody of IgG isotype within the cells; and detecting one or more microdrops containing a cell secreting an antibody of IgG isotype.
In some methods, the cells are cultured in the presence of an agent that stimulates isotype switching. In some methods, the capture reagent is an antibody to the IgG isotype. In some methods, the detecting comprises contacting the microdrops with a detection reagent that binds to the captured antibody at a different site than the capture reagent. In some methods, the detection reagent is an anti-idiotypic antibody. In some methods, the anti-idiotypic antibody is fluorescently labelled. Some methods further comprise isolating the microdrop that has captured the cell secreting the IgG antibody. In some methods, the population of cells are encapsulated in microdrops such that at least some microdrops encapsulate a plurality of cells; and the method isolates a microdrop that has captured a plurality of cells, one or more of which secretes IgG antibody. Some methods further comprise isolating the plurality of cells. Some methods further comprise encapsulating the plurality of cells in microdrops under conditions whereby at least some microdrops encapsulate a single one of the plurality of cells, wherein the microdrops include a capture reagent specific for antibodies of IgG isotype, whereby microdrops containing a cell secreting an antibody of IgG isotype capture the secreted antibody within the microdrops; and detecting one or more microdrops containing a cell secreting an antibody of IgG isotype.
The invention further provides methods for screening a population of cells for a subpopulation having a desired property. Such methods entail (a) encapsulating the population of cells in microdrops at a first ratio of average number of cells per occupied microdrop; (b) screening the encapsulated cells to identify a first subpopulation of microdrops encapsulated cells having the desired property; (c) isolating the cells from the microdrops resulting from the screening step; (d) encapsulating the cells from the screening step at a second ratio of average number of cells per occupied microdrop, the second ratio being smaller than the first ratio; (e) screening the encapulsated cells to identify a second subpopulation of microdrops encapsulating cells having the desired property.
Some methods further comprise repeating steps (c)-(e) at a further ratio of average number of cells per occupied microdrops to isolate a further subpopulation of microdrops.
Some methods further comprise isolating a microdrop encapsulating a single cell from the second subpopulation. In some methods, the subpopulation of cells having the desired property is less than 0.01% of the population of cells before the method is performed.
The invention further provides a kit for making microdrops. Such a kit comprises matrix component molecules linked to biotin, in a molar ratio of less than 0.85, preferably 0.01 to 0.2 and optionally 0.02 to 0.2 moles biotin per mole matrix component molecules. In some kits, the matrix molecules are agarose. Some kits further comprise instructions for using the kit to make microdrops. Some kits further comprise streptavidin and a capture molecule linked to second biotin molecules.